Institute for Bioengineering and Biosciences

Funder

Organizational Unit

Publications

Biophysical and biological properties of atypical sphingolipids : implications to physiology and pathophysiology

Publication . Da Cunha Branco Dos Santos, Tânia; Silva, Liana Casquinha da; Prieto, Manuel; Hornemann, Thorsten

1-deoxy-sphingolipids lack the C1-OH on their sphingoid base and present a cis ∆14-15 double bond instead of the canonical trans ∆4-5 double bond. Increased 1-deoxy-sphingolipid levels are directly correlated with the development and progression of hereditary sensory and autonomic neuropathy type 1 (HSAN1) and diabetes type 2. Different mechanisms have been proposed to explain the cytotoxicity of 1-deoxy-sphingolipids. However, these are highly dependent on the cell line studied and on the lipid concentration used, limiting the understanding of the mechanisms by which 1-deoxy-sphingolipids exert their patho-physiological roles. As for other sphingolipids, 1-deoxy-sphingolipids biological action might be related to the specific changes that each of the species cause on the biophysical properties of the membranes. Nonetheless, studies that comprehensively address the 1-deoxy-sphingolipids biophysical behavior are still scarce. Thereby, the goal of this study was to bring more insight into the biophysical impact of 1-deoxy-sphingolipids in both model and cellular membranes. Using complementary established fluorescence spectroscopy and microscopy methodologies and a multi-probe approach it was possible to conclude that: i) 1-deoxy-sphingolipids fail to form ordered domains as efficiently as canonical sphingolipids; ii) the presence, position and configuration of the sphingoid base double bond has a stronger influence on sphingolipids-induced changes on membrane biophysical properties than the structure of its C1 group; iii) external addition of 1-deoxy-sphingoid bases to living cells induce rapid changes in membrane fluidity in a sphingolipid structure dependent manner; iv) 1-deoxy-sphingolipids effects on membrane properties are specific and distinct from their canonical counterparts; v) endogenous elevation of 1-deoxy-sphingolipids due to mutations associated with HSAN1 development cause significant changes in the fluidity of cell membranes in a mutation dependent manner. Overall, the results suggest that pathologically elevated levels of 1-deoxy-sphingolipids compromise the biophysical properties of the membranes, which might impair proper cell functioning leading to the development of pathological conditions.

2021-06Doctoral thesis

Open access

Modeling Rett syndrome with human patient-specific forebrain organoids

Publication . Gomes, Ana Rita; Fernandes, Tiago G.; Vaz, Sandra H.; Silva, Teresa P.; Bekman, Evguenia; Xapelli, Sara; Duarte, Sofia; Ghazvini, Mehrnaz; Gribnau, Joost; Muotri, Alysson R.; Trujillo, Cleber A.; Sebastião, Ana M; Cabral, Joaquim M.S.; Diogo, Maria Margarida

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.

2020Journal article

Open access

Generation and characterization of induced pluripotent stem cell line (IBBISTi004-A) from an Angelman syndrome patient carrying a class II deletion of the maternal chromosome 15q11.2-q13

Publication . Maranga, Carina; Pereira, Carolina; Raposo, Ana Cláudia; Vieira, Adriana A.; Duarte, Sofia; Bekman, Evguenia; Milagre, Inês; da Rocha, Simão T.

Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease.

2022Journal article

Open access

Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Publication . Arez, Maria; Eckersley-Maslin, Melanie; Klobuar, Tajda; von Gilsa Lopes, João; Krueger, Felix; Mupo, Annalisa; Raposo, Ana Cláudia; Oxley, David; Mancino, Samantha; Gendrel, Anne-Valerie; Jesus, Bruno Bernardes De; da Rocha, Simão T.

Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.

2022Journal article

Open access

In the flow of molecular miniaturized fungal diagnosis

Publication . Zolotareva, Maria; Cascalheira, Francisco; Caneiras, Catia; Bárbara, Cristina; Caetano, Diogo Miguel; Teixeira, Miguel Cacho

The diagnosis of fungal infections presents several challenges and limitations, stemming from the similarities in symptomatology, diversity of underlying pathogenic species, complexity of fungal biology, and scarcity of rapid, affordable, and point-of-care approaches. In this review, we assess technological advances enabling the conversion of cutting-edge laboratory molecular diagnostic methods to cost-effective microfluidic devices. The most promising strategies toward the design of DNA sequence-based fungal diagnostic systems, capable of capturing and deciphering the highly informative DNA of the pathogen and adapted for resource-limited settings, are discussed, bridging fungal biology, molecular genetics, microfluidics, and biosensors.

2024Journal article

Restricted access